Follicle-stimulating hormone (FSH) is a protein falling into the class of gonadotrophins. FSH is used in the treatment of infertility and reproductive disorders in both female and male patients.
In nature, FSH is produced by the pituitary gland. For pharmaceutical use, FSH may be produced recombinantly (rFSH), or it may be isolated from the urine of postmenopausal females (uFSH).
FSH is used in female patients in ovulation induction (OI) and in controlled ovarian hyperstimulation (COH) for assisted reproductive technologies (ART). In a typical treatment regimen for ovulation induction, a patient is administered daily injections of FSH or a variant (about 75 to 300 IU FSH/day) for a period of from about 6 to about 12 days. In a typical treatment regimen for controlled ovarian hyperstimulation, a patient is administered daily injections of FSH or a variant (about 150-600 IU FSH/day) for a period of from about 6 to about 12 days.
FSH is also used to induce spermatogenesis in men suffering from oligospermia. A regimen using 150 IU FSH 3 times weekly in combination with 2'500 IU hCG twice weekly has been successful in achieving an improvement in sperm count in men suffering from hypogonadotrophic hypogonadism [Burgues et al.; Subcutaneous self-administration of highly purified follicle stimulating hormone and human chorionic gonadotrophin for the treatment of male hypogonadotrophic hypogonadism. Spanish Collaborative Group on Male Hypogonadotrophic Hypogonadism; Hum. Reprod.; 1997, 12, 980-6].
FSH agonists/FSH mutants with increased half-lives have been developed by fusing the carboxyterminal peptide of hCG (CTP) to native recombinant human FSH (rhFSH). The CTP moiety consists of amino acids 112-118 to 145 with four O-linked glycosylation sites located at positions 121, 127, 132 and 138. U.S. Pat. No. 5,338,835 and U.S. Pat. No. 5,585,345 disclose a modified FSH β-subunit extended at the C-terminal Glu with the CTP moiety of hCG. The resulting modified analogue is stated to have the biological activity of native FSH, but a prolonged circulating half-life. U.S. Pat. No. 5,405,945 discloses that the carboxy terminal portion of the hCG beta-subunit or a variant thereof has significant effects on the clearance of CG, FSH, and LH.
U.S. Pat. No. 5,883,073 discloses single-chain proteins comprised of two α-subunits with agonist or antagonist activity for CG, TSH, LH and FSH. U.S. Pat. No. 5,508,261 discloses heterodimeric polypeptides having binding affinity to LH and FSH receptors comprising a glycoprotein hormone α-subunit and a non-naturally occurring β-subunit polypeptide, wherein the β-subunit polypeptide is a chain of amino acids comprising four joined subsequences, each of which is selected from a list of specific sequences. Klein et al. (2003) discloses a single chain analogue of FSH with an increased half-life, wherein the α- and beta-subunits are linked by an oligopeptide containing two N-linked glycosylation sites.
WO 01/58493 discloses 77 mutations that may be made in the α-subunit of FSH and 51 mutations that may be made in the beta-subunit of FSH in an attempt to improve the in vivo half-life of FSH. WO 01/58493 discloses that the mutant α- and β-subunits may be used individually (1 additional glycosylation site) or in combination (2 additional glycosylation sites). The 128 candidate mutants were identified by using 50 models of the 3D structure of FSH that were generated based solely on the structure of hCG and a sequence alignment of FSH and hCG despite only 32% identity between the beta-subunits of hCG and FSH. WO 01/58493 does not disclose the production or testing of any α- or β-subunits of FSH where a glycosylation site was introduced by site directed mutagenesis.
WO 05/020934 discloses the FSH mutant GM1, with mutations in both the alpha- and beta-subunits of FSH, including a mutation at H83N of the alpha-subunit and a double mutation at E55N/V57T of the beta-subunit.
Because of the importance of FSH in the treatment of fertility disorders, the provision of FSH or mutants of FSH of high purity and high specific activity is desirable. FSH treatment requires repeated injections. Highly purified FSH preparations can be administered subcutaneously, permitting self-administration by the patient, thus increasing patient convenience and compliance.
Lynch et al. [The extraction and purification of human pituitary follicle-stimulating hormone and luteinising hormone; Acta Endocrinologica, 1988, 288, 12-19] describe a method for purifying human pituitary FSH. The method involves anion and cation exchange chromatography, immunoaffinity extraction and size exclusion chromatography. The method is said to result in pituitary FSH having a specific activity of 4,990 IU (immunoassay)/mg, with 16 IU/mg of LH. Protein content was determined either by dry weight or in solution by absorption at 280 nm (assuming that A2801cm for 1 g/l is equal to 1).
WO 98/20039 (IBSA Institut Biochimique SA) describes a process for the purification of human urinary FSH starting with urinary extracts called human menopausal gonadotrophins (hMG). The process uses ion-exchange chromatography on weakly basic anionic exchange resins of the DEAE type followed by affinity chromatography on resin having an anthraquinone derivative as a ligand. The process is said to yield urinary FSH free from LH and having a specific activity of 6,870 IU (immuno-assay)/mg. Protein content was determined by assuming that a water solution of 1 mg/ml of protein has an optical density of 0.62 at 277 nm, in quartz cuvettes with a 1 cm path length.
WO 00/63248 (Instituto Massone SA) describes a process for the purification of gonadotrophins, including FSH, from human urine. The process involves the following steps: ion exchange chromatography with a strong cationic resin of the type sulphopropyl, ion exchange chromatography with a strong anionic resin, and hydrophobic interaction chromatography (HIC). A FSH preparation having a specific activity of 8,400 IU/mg (Steelman-Pohley method: Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotrophin; Endocrinology; 1953, 53, 604-616) and less than 1 IU LH (rat seminal vesicle weight gain method: Van Hell H, Matthijsen R & G A Overbeek; Acta Endocrinol, 1964, 47, 409) biological activity per 75 IU FSH is reportedly obtained. Protein content was performed by the Lowry method [O. H. Lowry et al., J. Biol. Chem., 1951, 193, 265].
U.S. Pat. No. 5,990,288 (Musick et al.) describes a method for purifying FSH from biological samples, such as human pituitary glands or human post-menopausal urine. The process uses cation exchange chromatography on Fractogel EMD SO3-650M, followed by dye affinity chromatography on Mimetic Orange 1 resin, followed by a step of hydrophobic interaction chromatography on Bakerbond Wide Pore HI-Propyl resin. The process is said to result in human pituitary FSH having a specific activity of 7,066 IU (immunoassay)/mg and less than 1 IU (immunoassay)/mg of LH, and a urinary FSH having a specific activity of 6,298 IU (immunoassay)/mg and less than 3 IU (immunoassay)/mg of LH. Protein content was determined by absorption at 280 nm (assuming that A2801cm for 1 g/l is equal to 1).
Chiba et al. [Isolation and partial characterisation of LH, FSH and TSH from canine pituitary gland; Endocrinol. J., 1997, 44, 205-218] describe a technique for purifying canine pituitary gonadotrophins, including FSH, using Concanavalin (Con) A affinity chromatography, hydrophobic interaction chromatography (HIC) and immobilized metal ion chromatography with Cu++. The resulting FSH is reported to have a specific activity of 2.17 IU/g protein using a radioreceptor assay for FSH for measuring biological activity and the BioRad protein assay kit (BioRad Laboratories CA USA) for determining protein content.
WO 88/10270 (Instituto di Ricerca Cesare Serono SPA) describes a method for purifying human FSH from urine. The process involves immunochromatography with FSH-specific immobilized monoclonal antibodies bound to Sepharose 4B by divinyl sulphone, followed by reverse phase HPLC. The resulting FSH is free of LH and other urinary proteins and has a specific activity of 6,200 IU/mg of lyophilised powder (Steelman-Pohley method). The preparation was the first FSH preparation to be suitable for subcutaneous administration, due to its purity.